The field of this invention is electrophoretic separation using polymer-containing media.
Capillary electrophoresis is one of the most widely used separation techniques in the biologically related sciences. It finds application in genetics, for DNA sequencing, single nucleotide polymorphism (xe2x80x9csnpxe2x80x9d) detection, identification of sequences, gene profiling, etc.; in drug screening, particularly high throughput drug screening, where the electrophoresis allows for the use of impure reagents, separation of entities that can interfere with detection of a signal, particularly an electromagnetic signal; for performing reactions by bringing together reactants and allowing for their automated separation, segregation, purification and analysis without manual intervention, and the like. Due to the highly efficient heat dissipation, capillary electrophoresis permits rapid and efficient separations of charged substances. Charged substances will be subject to two electromigrating forces under the influence of the applied electrical potential at both ends of the capillary. One is the electrophoretic mobility component, which depends on the charge and size of the entity and the electrical field strength. The other is the electroendosmotic flow (xe2x80x9cEOFxe2x80x9d) providing a fixed bulk velocity component, which drives both neutral species and ionic species, regardless of mobility, towards an electrode in relation to the charge on the wall of the capillary.
The magnitude of EOF is highly dependent on the surface charge and viscosity near the surface of the capillary. These properties are affected by the material of the capillary wall, the buffer or medium composition and the pH. In the case of a silica capillary, upon ionization the negatively charged inner wall attracts a layer of positive ions from the buffer. As these mobile ions flow toward the cathode under the influence of the electrical potential, the bulk solution also flows in this direction to maintain electroneutrality. Control and suppression of EOF improves the separation resolution in such situations as capillary zone electrophoresis, capillary isoelectrofocusing and capillary gel electrophoresis. Failure to suppress the EOF can result in inadequate separations, especially with nucleic acids and proteinaceous materials.
With the inception of microfabricated devices used as the structure to carry out electrophoresis inside a microchannel, particularly where the device is composed of plastic, plastics may vary as the amount of wall charge, the adverse effect of EOF may be amplified. Many plastic materials are attractive for manufacturing large numbers of microfabricated devices, since molds can be produced with intricate designs of interconnected channels and reservoirs. These molds may then be used for the precise fabrication of the microfabricated devices rapidly and inexpensively. Various materials which find use include the polymethacrylates and other acrylics, polycarbonates, dimethylsiloxanes, polyalkenes, etc. Each of these polymers will have a different surface chemistry.
Based on the experience with fused silica, the silanol Sixe2x80x94OH ionized from the silica surface produces a negative charged surface. A diversity of treatments has been developed for suppressing the EOF in silica capillaries to minimize the interaction between the species to be analyzed and the capillary inner wall. The underlying idea is to reduce the production of charges. With the development of plastic devices, new approaches are required to modify the plastic surface to diminish EOF. The EOF may be attributed to the plastic composition and the nature of the plastic surface, where the plastic surface is conditioned by the polymerization process and the forming or production conditions of the device.
To suppress both electro osmotic flow as well as DNA-capillary wall interactions, in DNA sequencing using electrophoresis, control of the capillary inner surface is required for hydrophobic sieving polymers, such as polyacrylamide and hydroxyethylcellulose. Both permanently covalent and dynamically temporal modification of the inner surface can be applied. However, the procedures to produce covalent coatings are often complicated and time-consuming and the coatings may be subject to chemical instability over extended use. In order to reduce DNA sequencing cost, improve the reproducibility and reliability, it is therefore desirable to replace the expensive and often unreliable covalent coatings with adsorbed coatings.
References of interest include U.S. Pat. Nos. 4,865,707; 5,112,460; 5,663,129; 5,832,785, and 5,840,338. References of interest include Karger, et al., Anal. Chem. 1998, 70, 3996-4003; Fung, et al., ibid, 1999, 71, 566-573; and Madabhushi, Electrophoresis, 1998, 19, 224-230.
Compositions and methods for performing capillary electrophoresis, particularly in polymer-substrate microfluidic devices, are provided employing a mixed uncrosslinked polymer composition, which serves as both a coating reagent and sieving media in a microfluidic device. The polymeric composition comprises a mixture of polymers, where the more hydrophilic polymers serve primarily as sieving polymers and the less hydrophilic polymers serve primarily as coating polymers. Of particular interest is the use of uncrosslinked polyacrylamide and N-substituted polyacrylamide with varying hydrophilicity as a result of varying the substituents on the amide nitrogen. Particularly, alkyl substituents are used of varying chain length. By appropriate selection, one can tune the hydrophilicity of one component for the best coating results, while controlling the other component(s) for the best sieving effect. The polymeric composition or the coating component thereof may be included in the media used in the microfluidic device channel prior to the electrophoretic process or the composition or only the sieving component may be included during the electrophoretic separation after application of the coating component with greatly enhanced separation of charged entities.